At least half of the victims of cardiovascular disease (CVD) are asymptomatic until the occurrence of a major vascular obstruction. Thrombus formation frequently results in the loss of peripheral blood circulation, endangering limb viability, and potentially in the loss of life. The risk of thrombus obstruction is particularly pronounced after blood vessel surgery, where thrombus formation and vascular occlusion are relatively common.
Thrombus formation involves a complex interaction of aggregated platelets and activated coagulation factors with the damaged vessel wall. Circulating platelets are nonadherent to normal endothelium or to each other, but when the endothelial lining of a vessel is broken, the platelets adhere to exposed subendothelial collagen. This is the first step in the formation of hemostatic plugs, and requires participation of a protein made by endothelial cells called the von Willebrand (vW) factor. The vW factor is found both in the vessel wall and in plasma, and binds during platelet adhesion to a receptor present on a glycoprotein of the platelet surface membrane. Next, platelets are activated in reactions initiated by collagen and by thrombin formed at the injury site. These stimuli activate phospholipase C, an enzyme that hydrolyzes the membrane phospholipid, phosphatidyl inositol triphosphate. Products of this reaction activate protein kinase C and also increase the calcium concentration of platelet cytosol. As a result, a series of progressive, overlapping events ensue. The platelets change shape and develop long pseudopods. A receptor is assembled on the platelet surface membrane, and fibrinogen and other adhesive proteins bind to this receptor causing platelets to stick to each other. Arachidonic acid is liberated from membrane phospholipids and undergoes oxidation to products that include prostaglandin H.sub.2 (PGH.sub.2), which serves as an important cofactor for collagen-induced platelet activation, and thromboxane A.sub.2 (TxA.sub.2), which can act itself as an additional platelet activator. The contents of platelets are secreted, including adenosine diphosphate (ADP) which can also stimulate platelet activation and recruit new platelets into the growing hemostatic plug.
Subsequent to platelet aggregation, fibrinogen in the circulating blood is converted to fibrin to physically tie the hemostatic platelet plug in place. The platelet surface undergoes a reorganization that exposes procoagulant phospholipids needed for enzyme/cofactor complexes of blood coagulation to form on the platelet surface. Secretion of platelet factor V from platelet s-granules provides a key component for one of the enzyme/cofactor complexes. As a result, thrombin is generated in increasing amounts on the platelet surface, and converts fibrinogen into fibrin with the formation of fibrin strands that radiate outward from aggregated platelets helping to secure the platelet plug to the site of injury. Additionally, a mechanism within the platelets is activated which results in contraction of platelet actinomycin. This compresses and consolidates the platelet plug, further securing it to the site of injury.
In the in vivo regulation of thrombus formation, platelet aggregation is mediated by the PGH.sub.2 derivative prostacyclin (PGI.sub.2). Prostacyclin is also a vasodilator and is believed to render the vessel lining inert to platelet interactions. Thus, TxA.sub.2 and PGI.sub.2 have opposing effects on platelet aggregation, and the degree of the physiological effect of each in the cardiovascular system on the regulation of thrombus formation is determined mainly by their quantitative balance (Bush, H. L., Jr. et al., "Favorable Balance of Prostacyclin and Thromboxane A.sub.2 Improves Early Patency of Human In Situ Vein Grafts," J. Vasc. Surg. 1:149-159, 1984; Coker, S. J. et al., "Thromboxane and Prostacyclin Release From Ischemic Myocardium In Relation To Arrhythmias," Nature 291:323-334, 1981; Hunter, G. C. et al., "Arterial Wall Thromboxane: Dominance After Surgery Predisposes to Thrombosis," J. Vasc. Surg. 1:314-319, 1984; and Zmuda, A., et al., "Experimental Atherosclerosis in Rabbits: Platelet Aggregation, Thromboxane A.sub.2 Generation and Antiaggregatory Potency of Prostacyclin," Prostaglandins 14:1035, 1977).
Therapeutic treatments to alter thrombus formation have focused mainly on inhibition of the aggregation response. The most widely accepted agent for this purpose is acetylsalicylic acid (ASA), or aspirin. In the arachidonic acid cascade, aspirin acts as a cyclooxygenase inhibitor, blocking the conversion of arachidonic acid to the PGH.sub.2 precursor prostaglandin G.sub.2 (PGG.sub.2). Since PGG.sub.2 is a precursor to both TxA.sub.2 and PGI.sub.2, aspirin blocks both the aggregation inducing and aggregation inhibiting effects of these factors, respectively. As an antithrombotic agent, aspirin has had varying degrees of success. Although minimal amounts of aspirin are required for platelet inhibition, most of the clinical experience relates to relatively large doses. The nonselective, potent inhibition of high-dose aspirin on both TxA.sub.2 and PGI.sub.2 has caused investigators to consider the theoretical advantage of the use of low-dose therapy. Preferred inhibition of proaggregatory TxA.sub.2 in humans has been limited to single-dose aspirin administration or short-term cumulative effect. It has been further found that the degree and duration of aspirin's beneficial effect is highly dependent on each subject's inherent thrombotic potential (see Zammit, M. et al., "Aspirin Therapy in Small Caliber Arterial Prostheses; Long Term Experimental Observations," J. Vase. Surg. 1(6 ):839-851, 1984). For many individuals, aspirin has little or no discernable antiaggregatory activity, and is not effective in reducing a predisposition for thrombus formation.
To overcome some of the problems associated with aspirin therapy, it has been previously proposed to utilize certain thromboxane synthetase inhibitors (TSIs), which inhibit the formation of proaggregatory TxA.sub.2 without interfering with the formation of antiaggregatory PGI.sub.2. Various imidazole derivatives have been proposed for this purpose, for use either alone or in connection with low dose aspirin therapy. See, for example, Kaplan, S. et al., "A New Combination Therapy for Selective and Prolonged Antiplatelet Effect: Results in the Dog," Stroke 17:450-454 (1986). It has also been suggested that zinc ions exhibit an inhibitory activity toward collagen-induced platelet aggregation and serotonin release in vitro (Chapvil, M. et al., "Inhibitory Effect of Zinc Ions on Platelet Aggregation and Serotonin Release Reaction," Life Sciences (16):561-572, 1975) and toward platelet activating factor (PAF) in vitro (Arch. Biochem. Biophys. 272(2):466-475 (1989); Arch. Biochem. Biophys. 260(2):841-846, 1988), and that hydroxyurea, citric acid and ascorbic acid inhibit plant lipoxygenase activity in vitro (Bekheet, I.A. et al., " The Effect of Some Inhibitors on the Activity of Lipoxygenase," Alex. Sci. Exch. 7(3):389-398, 1986).
Other therapeutic treatments to alter thrombus formation have involved a reduction in fibrinogen levels, since fibrinogen levels have been shown to be significantly related to cardiovascular risk factors, including blood viscosity, coagulation and fibrinolysis (see Kannel, W. B. et al., "Fibrinogen and Risk of Cardiovascular Disease --The Framingham Study," JAMA 258 (9):1183-1186, 1987; Wilhelmsen, L. et al., "Fibrinogen as a Risk Factor for Stroke and Myocardial Infarction," N. Eng. J. Med. 311 (8):501-504 (1984); and Stone, M. C. et al., "Plasma Fibrinogen --a Major Coronary Risk Factor," J. Roy. Col. Gen Prac. 35: 565-569, December, 1985).
Plasminogen activators, such as streptokinase and tissue plasminogen activator, directly reduce systemic fibrinogen levels and have been administered by intracoronary, intravenous and/or intramuscular injection for the acute phase treatment of coronary disorders, such as ischemic stroke and coronary thrombosis. In addition, fibrinogen degrading agents such as ancrod, a viper venom enzyme isolated from Agkistrodon rhodostoma, the Malaysian pit viper, have been shown to have anticoagulative and thrombolytic activity when administered by intravenous infusion (see, for example, Hossman, V. et al., "Controlled Trial of Ancrod in Ischemic Stroke," Arch. Neurol. 40:804-808, 1983; and Apprill, P. G., et al., "Ancrod Decreases the Frequency of Cyclic Flow Variations and Causes Thrombolysis Following Acute Coronary Thrombosis," Am. Heart J. 113(4):898-906, 1987). While plasminogen activators and ancrod have been shown to have some degree of efficacy when administered IV or IM in the acute phase treatment of thrombotic disorders, they are inappropriate for long term, routine administration in the chronic prophylaxis or treatment of cardiovascular disease.